The structure of the box C/D enzyme reveals regulation of RNA methylation

Lapinaitė, Audronė; Simon, Bernd; Skjærven, Lars; Rakwalska‐Bange, Magdalena; Gabel, Frank; Carlomagno, Teresa

doi:10.1038/nature12581

Cited by 157 publications

(166 citation statements)

References 45 publications

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“…SNORDs associate with fibrillarin, NOP56, NOP58, and NHP2L1 to form a canonical snoRNP (3). In this complex, the methyltransferase fibrillarin catalyzes 2′-O-methylation of the targeted ribose residue (6). We noted that the nuclear supernatant contained small amounts of the SNORD-associated proteins NHP2L1, NOP56, and NOP58.…”

Section: Resultsmentioning

confidence: 99%

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Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

Falaleeva

Pagès

Matuszek

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

165

152

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C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2′-Omethylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex, SNORD27 regulates the alternative splicing of the transcription factor E2F7 pre-mRNA through direct RNA-RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously "silent" exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes.alternative splicing | gene regulation | snoRNAs | pre-mRNA processing S mall nucleolar RNAs (snoRNAs) are 60-to 300-nt-long noncoding RNAs that accumulate in the nucleolus. Based on conserved sequence elements, snoRNAs are classified as C/D box small nucleolar RNAs (SNORDs) or H/ACA box snoRNAs (SNORAs). SNORDs contain sequence elements termed C (RUGAUGA) and D (CUGA) boxes, usually present in duplicates (C′ and D′ boxes), and up to two antisense boxes that hybridize to the target RNA (1). In humans, SNORDs are usually derived from introns. After the splicing reaction, introns are excised as lariats, which are then opened by the debranching enzyme and subsequently degraded. Intronic SNORDs escape this degradation by forming a protein complex that consists of non-histone chromosome protein 2-like 1 (NHP2L1, 15.5K, SNU13), nucleolar protein 5A (NOP56), nucleolar protein 5 (NOP58), and fibrillarin (2-4). The SNORD protein complex forms through the entry of the snoRNA and fibrillarin to a complex containing NHP2L1, NOP58, and at least five assembly factors (5). The SNORD acts as a scaffold for the final protein complex formation and also controls recognition of other RNAs using the antisense boxes. The antisense boxes recognize sequences in rRNA, resulting in the fifth nucleotide upstream of the D or D′ box being 2′-O-methylated by fibrillarin (1). Structural studies indicate that the active form of SNORDs is dimeric (6).The conserved overall structure of SNORDs allows the identification of their putative target RNA binding sites. However, numerous SNORDs without obvious target RNAs have been identified (7-10) and are termed "orphan snoRNAs." Genome-wide deep sequencing experiments identified shorter but stable SNORD fragments that were found in all species tested, ranging from mammals to the protozoan Giardia lamblia (11) and EpsteinBarr virus (12). Fragments longer than 27 nt generated by SNORDs will ...

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Section: Resultsmentioning

confidence: 99%

“…The antisense boxes recognize sequences in rRNA, resulting in the fifth nucleotide upstream of the D or D′ box being 2′-O-methylated by fibrillarin (1). Structural studies indicate that the active form of SNORDs is dimeric (6).…”

mentioning

confidence: 99%

Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

Falaleeva

Pagès

Matuszek

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

165

152

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“…In the proposed RNA-swapped model (34,35), the spacer regions of C/D RNAs are placed between two Nop5 dimers and mediate dimerization of the RNP (Fig. 3D).…”

Section: Discussionmentioning

confidence: 99%

“…The di-RNP structure has been analyzed by low-resolution approaches (34,35), but the key information about the organization of C/D RNA cannot be determined directly from these studies. Without invoking the unconventional RNA-swapped model, the di-RNP could be accounted for simply by self-association of the mono-RNP through protein-protein interaction.…”

Section: Discussionmentioning

confidence: 99%

“…This mechanism is incompatible with the RNA-swapped model for dimeric C/D RNP. C/D RNA has proposed to be swapped between two different protein complexes in the di-RNP (34)(35)(36)(37)(38), rather than associating with the same Nop5 dimer as shown in the mono-RNP structure (30). It also has been argued that the RNA-swapped di-RNP model is advantageous for accommodating long guide-substrate duplexes compared with the mono-RNP model.…”

Section: Significancementioning

confidence: 99%

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Box C/D guide RNAs recognize a maximum of 10 nt of substrates

Yang

Lin

2016

Proc. Natl. Acad. Sci. U.S.A.

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Box C/D RNAs guide site-specific 2′-O-methylation of RNAs in archaea and eukaryotes. The spacer regions between boxes C to D′ and boxes C′ to D contain the guide sequence that can form a stretch of base pairs with substrate RNAs. The lengths of spacer regions and guide-substrate duplexes are variable among C/D RNAs. In a previously determined structure of C/D ribonucleoprotein (RNP), a 12-nt-long spacer forms 10 bp with the substrate. How spacers and guide-substrate duplexes of other lengths are accommodated remains unknown. Here we analyze how the lengths of spacers and guide-substrate duplexes affect the modification activity and determine three structures of C/D RNPs assembled with different spacers and substrates. We show that the guide can only form a duplex of a maximum of 10 bp with the substrate during modification. Slightly shorter duplexes are tolerated, but longer duplexes must be unwound to fit into a capped protein channel for modification. Spacers with <12 nucleotides are defective, mainly because they cannot load the substrate in the active conformation. For spacers with >12 nucleotides, the excessive unpaired sequences near the box C/C′ side are looped out. Our results provide insight into the substrate recognition mechanism of C/D RNA and refute the RNAswapped model for dimeric C/D RNP.B ox C/D RNAs are a large family of noncoding RNAs conserved in archaea and eukaryotes (1-3). The majority of these RNAs function as guides in site-specific 2′-O-methylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (4-7). A few special members, including U3 and U14, small nucleolar RNAs are required for pre-rRNA processing and ribosome assembly. One of the most abundant modifications in rRNAs, 2′-O-methylated nucleotides cluster in functionally important regions and are believed to fine-tune ribosome structure and function (8)(9)(10)(11).Box C/D RNAs have a bipartite structure and contain the C (RUGAUGA; R is a purine) and D (CUGA) box motifs at two ends and the related C′ and D′ box motifs in the internal regions. Each pair of boxes C and D combine into a characteristic kink turn (K-turn) or K-loop structure (12-15). The spacer regions located between boxes D and C′ (D spacer) and between boxes D′ and C (D′ spacer) harbor the guide sequence. The guide sequence can potentially form 10-21 bp with complementary sequences in substrate RNAs and select the fifth nucleotide apart from box D′/D for modification (5).To form a functional ribonucleoprotein (RNP) enzyme, each C/D RNA associates with four core proteins [Snu13, fibrillarin (Nop1 in yeast), Nop56, and Nop58] in eukaryotes (16-23) and with three proteins (L7Ae, fibrillarin, and Nop5) in archaea (24). The homologous eukaryotic Nop56 and Nop58 proteins are replaced by a single protein, Nop5, in archaea. Fibrillarin is the catalyst that transfers a methyl group from S-adenosylmethionine to the target ribose (21, 25). The RNA-guided methylation activity has been reconstituted for archaeal C/D RNPs (24), but not yet for eukaryotic complexes (26).Structural st...

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RNAModification

Motorin

2015

Encyclopedia of Life Sciences

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Naturally occurring ribonucleic acids (RNAs) contain over 150 chemically altered nucleosides formed by enzymatic modification of the primary RNA transcript during the complex tRNA maturation process. This post‐transcriptional RNA modification is a universally conserved and highly complex metabolic process in the living cell. Cellular RNAs are modified by pure protein stand‐alone enzymes, as well as by s(sno)RNP complexes containing guide s(sno)RNAs. Modified nucleotides present in RNA play an important role in stabilisation of 2D and 3D structures of these molecules, as well as in the fine‐tuning of numerous interactions between RNAs itself and with RNA‐binding protein partners. Modification also protects RNAs against nucleolytic degradation and improves their performance in different interactions in which various RNAs are involved. For example, modifications present in the tRNA anticodon loop are crucially important for correct mRNA decoding during the protein synthesis on the ribosome. Recent progress in the field points out the regulatory character of RNA modification. This emerging concept of ‘RNA‐epigenetics’ supplies the additional level to the regulation of gene expression. This regulation (and deregulation) of RNA‐modification machineries is a basis for some important human pathologies. Key Concepts Cellular RNAs are post‐transcriptionally modified in all life kingdoms RNA modification alters physico‐chemical properties of nucleotides, including their conformation, polarity, hydrophobicity, chemical reactivity and base‐pairing interactions RNA modification is performed by highly specific and regulated enzymatic mechanisms involving pure protein enzymes and catalytic RNA–protein complexes (RNPs) RNA modification is important for regulation of gene expression Transcription‐wide RNA modification is dynamic and regulated cellular process Deregulation of RNA modification may lead to important human pathologies

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The structure of the box C/D enzyme reveals regulation of RNA methylation

Cited by 157 publications

References 45 publications

Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

Box C/D guide RNAs recognize a maximum of 10 nt of substrates

RNAModification

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